Clinical studies report that children born with severe hyperhomocysteinemia (HHcy), due to deficiency in cystathionine-?-synthase (CBS) gene, resulting in an elevation in plasma homocysteine (Hcy) levels, develop skeletal malformations with weaker bone. Although mitochondrial dysfunction has been involved in bone pathology, altered mitochondrial (Mito) epigenetics and dynamics contributing to frailer bone have not been studied. The long term goal of this project is to understand the mechanisms of mito-epigenetic remodeling and changes in bone structure/function during genetic HHcy. Alterations in mitochondrial dynamics disturb bone cell (osteoclast/ osteoblast) differentiation and matrix metabolism. Studies have confirmed that type-I collagen (coll-1), a dominant protein of the organic bone matrix, is homocysteinylated (N-Hcy-coll-1) during HHcy. Our preliminary data suggest that genetic HHcy affects mito-epigenetic remodeling by altering mito-redox stress, mito-DNA methylation / hydroxymethylation, fission-fusion and cause N-Hcy-coll-1 cross linking. These processes cumulatively disrupt bone matrix, bone density, bone blood flow and lead to mitophagy accompanying with significant bone loss. Interestingly, hydrogen sulfide (H2S, an anti-redox stress and Hcy lowering agent) mitigates bone damage. The central hypothesis of this proposal is that HHcy contributes to bone-matrix inferiority through N-Hcy-coll-1, in part, by increasing mito- epigenetic remodeling and altering mitochondrial dynamics. However, treatment with H2S ameliorates HHcy-induced bone abnormalities. We will test this hypothesis by following three specific Aims: Specific Aim # 1: To determine whether the HHcy instigates mito-epigenetic remodeling and DNA methylation, in part, by altering mitochondrial fission and fusion, and whether H2S mitigates mito-epigenetic remodeling. Specific Aim # 2: To determine whether the HHcy homocysteinylates bone type-I collagen, activates MMP-1,-3,-13, disrupts bone matrix (MMP/TIMP axis; collagen/elastin ratio) and H2S ameliorates these changes. Specific Aim # 3: To determine whether the HHcy alters bone mineral density, bone mineral content, biomechanical load-bearing capacity, bone blood flow and disrupts osteoblast/osteoclast differentiation and whether H2S alleviates these alterations.